The post-translational modification of serine and threonine residues on proteins by O-linked N-acetylglucosamine (O-GlcNAc), is increasingly recognized as being as abundant as phosphorylation and playing a similarly important role in regulating diverse cell functions including cell cycle, transcription, protein degradation, mitochondrial function, autophagy, circadian rhythm, and cell survival. Activation of the hexosamine biosynthesis pathway, which regulates protein O-GlcNAcylation has been associated with increased lifespan in both C. elegans and mice. In the heart dysregulation in O-GlcNAc homeostasis has been linked to different disease processes including cardiac hypertrophy, heart failure, the adverse effects of diabetes and age-related changes in cardiomyocytes. On the other hand, acute increases in O-GlcNAc levels promotes resilience of cardiomyocytes in response to ischemia/reperfusion injury. We have shown that O-GlcNAcylation regulates physiological processes that are key to maintaining healthy cardiomyocytes. Our preliminary data also show that fasting increases O-GlcNAc levels in the heart, strongly supporting a physiological role for protein O- GlcNAcylation. Despite the association of O-GlcNAc with cardiac disease our knowledge of the basic regulatory mechanisms in the normal healthy heart remains limited, there is growing evidence that O-GlcNAc cycling contributes to the regulation of normal physiological processes in a healthy heart. A lack of understanding of the fundamental biology underlying how O-GlcNAc regulates normal cardiomyocyte function represents a major gap in our knowledge and limits the potential for modulation of O-GlcNAc homeostasis in the treatment of cardiac disease. Consequently, we believe that there is an urgent need to better understand the basic biology underlying the effects of O-GlcNAc on cardiomyocyte function. Therefore, we will test the hypothesis that protein O- GlcNAcylation plays an integral role in normal cardiomyocyte homeostasis contributing to cardiomyocyte resilience in response to physiological stimuli. We will test this hypothesis with two specific aims: 1) Determine how changes in O-GlcNAc levels influence the molecular and cellular responses of the heart to fasting; and 2) Determine how physiological stimuli influence the temporal and intracellular localization of OGT and OGA, and O-GlcNAc. We will use inducible cardiomyocyte specific mouse models to increase or decrease O-GlcNAc levels in the heart, genetically based O-GlcNAc FRET sensors, and fluorescently tagged OGT and OGA to determine the spatiotemporal dynamics of O-GlcNAc that occur in response to physiological stimuli. The successful completion of the proposed studies will yield significant new information on the role of O-GlcNAc in regulating normal cardiac physiology including its role in maintaining cardiomyocyte resilience. This application is directly responsive to PA-19-049 by focusing on improving our fundamental knowledge of the cardiac glycome and its ability to regulate cardiovascular biology. These findings will lay the foundation that will help further our understanding of this signaling pathway in cardiac health and resilience.